It is the aim of the proposed research to gain an understanding of how Streptomyces elaborate the nitrogen-nitrogen bonds which occur in two antibiotics. The antibiotics to be studied are streptozotocin and N-acetyl-6-diazo-5-oxy-L-norleucine (N-acetyl-DON) as representatives of compounds containing N-nitrosamide and diazo functionalities, respectively. These antibiotics are of clinical and/or experimental interest since they are known to act either as anticancer, as diabetogenic or as carcinogenic agents. Previous work in this laboratory has provided information on the origin of the carbon skeleton or streptozotocin and satisfactory conditions for the investigation of the N-acetyl-DON biosynthetic pathway have been explored. Further feeding experiments on the latter pathway will be concerned with the substantiation of the role of lysine in this biosynthesis. These experiments will be followed in the case of both antibiotics by detailed investigations of the origin of the linked nitrogen atoms using 15N labeled precursors and mass spectral and Fourier transform 15N nuclear magnetic resonance spectroscopic analysis to localize the labeled atoms in both antibiotics. Further studies will then be carried out at the enzymatic level to explore the biochemical steps and mechanisms involved in N-N bond biosynthesis. Special emphasis will be placed on the origin and the fate in the living cell of nitrous acid which is assumed to play a key role in these biosynthetic pathways. Concurrently, efforts will be made to investigate if antibiotic nonproducing mutants can be isolated from the relevant strains of Streptomyces and these mutants will be used to characterize the biosynthetic pathways genetically.